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The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.

Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, Bazett-Jones DP - J. Cell Biol. (2001)

Bottom Line: In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100.They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains.Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

ABSTRACT
The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

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Related in: MedlinePlus

FRAP of half-nucleus of 293 cells expressing GFP–Pml (A), GFP–Sp100 (B), or GFP–CBP (C). Cells were imaged before the bleaching (Pre-Bleached), immediately after the bleaching (Bleached, time = 0 s), and during fluorescence recovery at the indicated times. A box indicates the area bleached, corresponding to half the nucleus. The fluorescence intensity in the bleached and the unbleached regions was measured and expressed as a relative intensity where a value of one is equal intensity in both halves. The relative intensity over time is shown. Fluorescence recovery curves for CBP (D), Pml (E), and Sp100 (F) show the kinetics of redistribution of the different fluorescent proteins after bleaching. As a control, recovery of GFP alone was measured (G).
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Figure 3: FRAP of half-nucleus of 293 cells expressing GFP–Pml (A), GFP–Sp100 (B), or GFP–CBP (C). Cells were imaged before the bleaching (Pre-Bleached), immediately after the bleaching (Bleached, time = 0 s), and during fluorescence recovery at the indicated times. A box indicates the area bleached, corresponding to half the nucleus. The fluorescence intensity in the bleached and the unbleached regions was measured and expressed as a relative intensity where a value of one is equal intensity in both halves. The relative intensity over time is shown. Fluorescence recovery curves for CBP (D), Pml (E), and Sp100 (F) show the kinetics of redistribution of the different fluorescent proteins after bleaching. As a control, recovery of GFP alone was measured (G).

Mentions: To determine whether CBP is a mobile molecule in the nucleus and moves in or out of PML nuclear bodies, we measured its mobility using the FRAP technique. The approach uses a high intensity laser from a laser scanning microscope to irreversibly bleach a region in the nucleus of a cell expressing a GFP fusion protein (boxes in Fig. 3 represent the bleached region). After photobleaching, the cell is imaged at different times to follow the redistribution of the unbleached GFP fusion proteins. Because the bleaching process irreversibly eliminates fluorescence of the GFP without affecting the rest of the protein, it is possible to visualize the normal movement of the unbleached protein in the cell. The recovery of the fluorescence is a measure for the mobility of the protein.


The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.

Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, Bazett-Jones DP - J. Cell Biol. (2001)

FRAP of half-nucleus of 293 cells expressing GFP–Pml (A), GFP–Sp100 (B), or GFP–CBP (C). Cells were imaged before the bleaching (Pre-Bleached), immediately after the bleaching (Bleached, time = 0 s), and during fluorescence recovery at the indicated times. A box indicates the area bleached, corresponding to half the nucleus. The fluorescence intensity in the bleached and the unbleached regions was measured and expressed as a relative intensity where a value of one is equal intensity in both halves. The relative intensity over time is shown. Fluorescence recovery curves for CBP (D), Pml (E), and Sp100 (F) show the kinetics of redistribution of the different fluorescent proteins after bleaching. As a control, recovery of GFP alone was measured (G).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198823&req=5

Figure 3: FRAP of half-nucleus of 293 cells expressing GFP–Pml (A), GFP–Sp100 (B), or GFP–CBP (C). Cells were imaged before the bleaching (Pre-Bleached), immediately after the bleaching (Bleached, time = 0 s), and during fluorescence recovery at the indicated times. A box indicates the area bleached, corresponding to half the nucleus. The fluorescence intensity in the bleached and the unbleached regions was measured and expressed as a relative intensity where a value of one is equal intensity in both halves. The relative intensity over time is shown. Fluorescence recovery curves for CBP (D), Pml (E), and Sp100 (F) show the kinetics of redistribution of the different fluorescent proteins after bleaching. As a control, recovery of GFP alone was measured (G).
Mentions: To determine whether CBP is a mobile molecule in the nucleus and moves in or out of PML nuclear bodies, we measured its mobility using the FRAP technique. The approach uses a high intensity laser from a laser scanning microscope to irreversibly bleach a region in the nucleus of a cell expressing a GFP fusion protein (boxes in Fig. 3 represent the bleached region). After photobleaching, the cell is imaged at different times to follow the redistribution of the unbleached GFP fusion proteins. Because the bleaching process irreversibly eliminates fluorescence of the GFP without affecting the rest of the protein, it is possible to visualize the normal movement of the unbleached protein in the cell. The recovery of the fluorescence is a measure for the mobility of the protein.

Bottom Line: In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100.They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains.Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

ABSTRACT
The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

Show MeSH
Related in: MedlinePlus